A fluorescence-based high-throughput screening method for cytokinin translocation mutants.
A fluorescence-based high-throughput screening method for cytokinin translocation mutants.
复制标题
DOI：
10.1186/s13007-020-00676-4
复制
发表时间：
2020
期刊：
影响因子：
5.1
通讯作者：
Zhang K
中科院分区：
文献类型：
--
作者：
Zhang M;Ding B;Zhao J;Zhang P;Li Y;Yang G;Zhang K
细胞分裂素是植物生长发育和逆境反应所必需的一种植物激素。在过去的半个世纪里，细胞分裂素信号转导和代谢途径的研究取得了重大进展，但对细胞分裂素转位的机制知之甚少。拟南芥反应调节因子5(ARR5)是由细胞分裂素诱导的细胞分裂素信号转导途径中的A型反应因子，已被用作拟南芥内源细胞分裂素的报告基因。在这里，我们报道了一种基于荧光的高通量方法，使用由ARR5：：EGFP转基因植株产生的乙基甲基砜(EMS)突变文库来筛选细胞分裂素易位突变体。利用发光成像系统(LIS)对根中绿色荧光蛋白(GFP)信号增强的幼苗进行大规模筛选，获得根中细胞分裂素过量积累的突变体。筛选出的突变体在荧光显微镜下进行鉴定，然后进行表型分析。通过这种方法，我们获得了12个根中GFP信号增强的突变体，并进一步发现其中3个突变体在气生组织中显示GFP信号降低。其中两个突变体被鉴定为AtABCG14等位基因突变体，它们在根合成的细胞分裂素的长距离运输方面存在缺陷。我们提供了一种筛选细胞分裂素转位、分布或信号转导缺陷突变株的策略。该策略可用于建立一种系统，用于使用荧光报告程序筛选在其他激素转运体或信号组件上存在缺陷的突变体。
Cytokinins are one kind of phytohormones essential for plant growth, development and stress responses. In the past half century, significant progresses have been made in the studies of cytokinin signal transduction and metobolic pathways, but the mechanism of cytokinin translocation is poorly understood. Arabidopsis (Arabidopsis thaliana) response regulator 5 (ARR5) is a type-A response factor in cytokinin signaling which is induced by cytokinins and has been used as a reporter gene for the endogenous cytokinins in Arabidopsis. Here, we report a fluorescence-based high-throughput method to screen cytokinin translocation mutants using an ethyl methyl sulfone (EMS) mutagenesis library generated with ARR5::eGFP transgenic plants. The seedlings with enhanced green fluorescent protein (GFP) signal in roots were screened in a luminescence imaging system (LIS) in large scale to obtain mutants with over-accumulated cytokinins in roots. The selected mutants were confirmed under a fluorescence microscopy and then performed phenotypic analysis. In this way, we obtained twelve mutants with elevated GFP signal in the roots and further found three of them displayed reduced GFP signal in the aerial tissues. Two of the mutants were characterized and proved to be the atabcg14 allelic mutants which are defective in the long-distance translocation of root-synthesized cytokinins. We provide a strategy for screening mutants defective in cytokinin translocation, distribution or signaling. The strategy can be adapted to establish a system for screening mutants defective in other hormone transporters or signaling components using a fluorescence reporter.
登录
查看更多内容
DOI：
10.1073/pnas.0900060106
发表时间：
2009-03-17
影响因子：
11.1
作者：
Ruzicka, Kamil;Simaskova, Maria;Benkova, Eva
通讯作者：
Benkova, Eva
影响因子：
7.2
作者：
Beveridge, CA;Murfet, IC;Rameau, C
通讯作者：
Rameau, C
影响因子：
18
作者：
Osugi, Asami;Kojima, Mikiko;Sakakibara, Hitoshi
通讯作者：
Sakakibara, Hitoshi
影响因子：
11.6
作者：
Gillissen, B;Bürkle, L;Frommer, WB
通讯作者：
Frommer, WB
影响因子：
7.2
作者：
Clough, SJ;Bent, AF
通讯作者：
Bent, AF